Novel therapeutic use of 1,6-dimethyl-8b-hydroxymethyl-10a-methoxyergoline

ABSTRACT

The invention concerns the use of 1,6-dimethyl-8β-hydroxymethyl-10α-methoxyergoline for preventing and/or treating motor neuron diseases.

[0001] This application is a division of U.S. application Ser. No.09/863,610, filed May 23, 2001, which is a continuation of Internationalapplication No. PCT/FR99/02,868, filed Nov. 22, 1999; which claims thebenefit of priority of French Patent Application No. 98/14,792, filedNov. 24, 1998.

[0002] The present invention relates to the use of1,6-dimethyl-8β-hydroxymethyl-10α-methoxyergoline in the preventionand/or treatment of motor neuron diseases.

[0003] 1,6-Dimethyl-8β-hydroxymethyl-10α-methoxy-ergoline or1-methyl-10α-methoxy-9,10-dihydrolysergol is one of the metabolites ofnicergoline (F. ARCAMONE et al., Biochem. Pharmacol., 21 (16), 2205-2013(1972)). Like nicergoline, but to a lesser degree, this compoundexhibits α1-adrenergic and 5HT1a-serotonergic properties. It is alsouseful as an intermediate for preparing nicergoline (French Patent No.2,616,788).

[0004] It has now been found that1,6-dimethyl-8β-hydroxymethyl-10α-methoxyergoline increases the survivalof motor neurons and can therefore be used in preventing and/or treatingmotor neuron diseases.

[0005] Motor neuron diseases include, in particular, amyotrophic lateralsclerosis, progressive spinal muscular atrophy, infantile muscularatrophy and primary lateral sclerosis.

[0006] In the presence of trophic support supplied by the neurotrophicfactors BDNF or GDNF, motor neuron cultures are composed of broad andhomogeneous neurons with long branched axons. However, the motor neuronsdie by apoptosis if the culture is performed in the absence of trophicsupport.

[0007] The effect of 1,6-dimethyl-8β-hydroxymethyl-10α-methoxyergolinehas therefore been determined in a degeneration model which is inducedby depriving cultured motor neurons of neurotrophic factor.

[0008] Furthermore, astrocytes play a major role in controlling andmaintaining an environment which is suitable for motor neuron survival.

[0009] 1,6-Dimethyl-8β-hydroxymethyl-10α-methoxy-ergoline has thereforealso been tested on a co-culture of motor neurons and astrocytes.

[0010] The following protocols were employed:

[0011] MOTOR-NEURON-ENRICHED CULTURES:

[0012] The motor-neuron-enriched cultures are prepared using thecentrifugation method which was described by R. L. SCHNAAR and A. E.SCHAFFNER, J. Neurosci., 1, 204-217 (1981) and modified by W. CAMU andC. E. HENDERSON, J. Neurosci. Methods, 44, 59-70 (1992). The motorneurons are spread, at a density of 2500 cells per plate, on 35 mmculture plates which have previously been coated with laminin/ornithinein accordance with the method of A. G. ESTEVEZ et al., J. Neurosci., 18(3), 923-931 (1998). The cultures are then maintained in L15 medium(GIBCO BRL) containing sodium bicarbonate (22 mM), conalbumin (0.1mg/ml), putrescine (0.1 mM), insulin (5 μg/ml), sodium selenite (31 mM),glucose (20 mM), progesterone (21 nM), penicillin (100 IU/ml) andstreptomycin (100 μg/ml).

[0013] The motor neurons which are thus obtained consist of large (25-30μm) and homogeneous neurons which possess long branched axons. More than70% of the cells are immunoreactive for the neurotrophin p75 receptorand the Islet ½ markers for spinal motor neurons. Approximately 70% ofthe motor neurons die by apoptosis 24 hours after the spreading if theculture is performed in the absence of a trophic factor.

[0014] CULTURES OF SPINAL CORD ASTROCYTES:

[0015] The astrocytes are obtained from young, one-day-old rats usingthe slightly modified method of R. P. SANETO and J. DE VELLIS asdescribed in Neurochemistry a practical approach (A. J. TURNER and H. A.St JOHN) IRL Press, Oxford-Washington D.C., pp. 27-63. The spinal cordsare dissected out under sterile conditions and freed of meninges anddorsal ganglia. Five to ten spinal cords are transferred into PBS(phosphate buffered saline) and cut before being incubated at 37° C. for25 minutes in PBS to which 0.25% trypsin has been added. The enzymatictreatment is stopped by adding 10 ml of Dulbecco's modified Eagle medium(DMEM), to which 10% fetal calf serum (FCS) has been added, and thecells are collected by centrifugation. Another step of mechanicaldissociation is performed using the end of a 1 ml pipette. The cells arespread, at a density of 1.5-2×10⁶ cells per 25 cm² of culture medium, inDMEM containing 10% FCS. After 2 days in vitro, the cultures are fedevery day. When a visible monolayer of cells has been completed, thecultures are stirred at 250 rpm for 48 hours and, on the following day,the monolayers are treated with cytosine arabinoside (10⁻⁵ M) for 48hours. The astrocyte monolayers are then amplified to a density of fiveon 35 mm culture plates for initially 25 cm² culture flasks.

[0016] The spinal astrocyte cultures consist, to an extent of more than98%, of flat, polygonal cells which are immunoreactive for the glialfibrillar acid protein (GFAP). The monolayers are exposed to the productto be tested and then incubated with the motor neuron medium in order toobtain a conditioned culture medium. This medium is transferred andtested at different dilutions in order to determine its effects onneuronal survival.

[0017] IMMUNOCHEMISTRY

[0018] The cells are fixed in 4% paraformaldehyde and 0.1%glutaraldehyde in PBS (pH 7.4 and at 4° C. for 15 minutes) and in a coldmethanolic solution. The cultures are then washed and the nonspecificsites are blocked with 10% goat serum and 2% bovine serum albumin (BSA)in PBS and treated for immunochemistry using antibodies against the p75weak affinity neurotrophin receptor or a 200 kD neurofilament protein(Amersham) using the manufacturer's instructions and applying theavidin-biotin-DAB/hydrogen peroxide enhancement reaction.

[0019] TREATING THE ASTROCYTES WITH1,6-DIMETHYL-8β-HYDROXYMETHYL-10α-METHOXYERGOLINE

[0020] The astrocytes are treated with1,6-dimethyl-8β-hydroxymethyl-10α-methoxyergoline in the followingmanner: the product is dissolved in methanol, sterilized by filtrationand used immediately after preparation. The treatment which is appliedto the enriched motor neuron cultures is effected by adding aliquots ofsolutions of the products to be tested to the L15 medium by spreading.The astrocyte monolayers are exposed to the vehicle or to the solutionsof the compound to be tested for 24 hours and at differentconcentrations. The astrocyte monolayers are washed 3 times with DMEMand incubated with complete L15 medium. The astrocyte-conditioned mediumis recovered 24 hours later and centrifuged at 1800 g for 15 minutes andused immediately or stored at −70° C. for a maximum of 2 weeks withoutloss of trophic activity.

[0021] COUNTING THE CELLS AND STATISTICAL ANALYSIS

[0022] The cells which are immunoreactive for neurofilaments and whichexhibit axons which are longer than the diameters of the cells areregarded as being viable motor neurons. The number of motor neurons isestimated by counting the labelled cells in an area of 0.4-1 cm² under amicroscope which enlarges 200 times. In all cases, the values areexpressed as a percentage of the number of motor neurons which arepresent in cultures maintained using trophic factors. The experimentsare carried out at least 3 times.

[0023] The statistical analyses are performed using the Student's test(t test).

[0024] The following results were obtained:

[0025] 1-Effect of 1,6-dimethyl-8β-hydroxymethyl-10α-methoxyergoline onneuronal survival in astrocyte/motor neuron co-cultures: % of labelledmotor neurons All the motor Very large motor neurons neurons Vehiclealone  100 ± 9.4 100 ± 2.8 1,6-Dimethyl-8β- hydroxymethyl-10α-methoxyergoline 0.1 μM   123 ± 9.3** 122.6 ± 8.2* 1 μM 158.9 ± 32*   117± 5.6* 10 μM 137.5 ± 19* ND

[0026] These results demonstrate that1,6-dimethyl-8β-hydroxymethyl-10α-methoxyergoline (0.1-1 μM) preventsthe neuronal death of the co-cultures and increases the number of largemotor neurons.

[0027] 2-Effect of 1,6-dimethyl-8β-hydroxymethyl-10α-methoxyergoline onthe neurotrophic activity of the motor neurons which is produced by thespinal astrocytes: Motor neuron survival in % Dilutions of theastrocytes in the conditioned medium 1:50 1:10 1:4 Vehicle alone 36.9 ±3.2 45.4 ± 4.2 59.9 ± 8 1,6-Dimethyl-8β- 49.6 ± 5.3** 74.8 ± 6.3** 90.6± 4.9** hydroxymethyl 10α-methoxy-- ergoline

[0028] These results demonstrate that1,6-dimethyl-8β-hydroxymethyl-10α-methoxyergoline stimulates the motorneuron trophic activity which is produced by the spinal astrocytemonolayers.

[0029] 3-Effect of 1,6-dimethyl-8β-hydroxymethyl-10α-methoxyergoline onco-cultures of astrocyte monolayers and motor neurons Motor neuronsurvival in % Concentration of the tested product 1 μM 100 nM 10 nM 1 nMVehicle 43.9 ± 4 alone 1,6- 68.7 ± 8** 57.4 ± 4** 48.9 ± 3** 42.5 ± 4Dimethyl- 8β-hydroxy- methyl-10α- methoxy-ergoline

[0030] These results demonstrate the efficacy of1,6-dimethyl-8β-hydroxymethyl-10α-methoxyergoline in motor neurontrophic stimulation by the astrocytes.

[0031] 4-Neurotrophic-like effect of1,6-dimethyl-8β-hydroxymethyl-10α-methoxyergoline on neuronal death inmotor-neuron-enriched cultures and in the absence of trophic factor:

[0032] 1,6-Dimethyl-8β-hydroxymethyl-10α-methoxy-ergoline increases thesurvival of trophic factor-deprived motor neurons by 60% (p<0.001),while neuronal arborization and cell morphology remain well preserved.

[0033] These results suggest that1,6-dimethyl-8β-hydroxymethyl-10α-methoxyergoline exerts a neurotrophiceffect on this degeneration model, which is induced by deprivingcultured motor neurons of trophic factor, and, furthermore, stimulatesthe ostracise which produce this neurotrophic factor.

[0034] The present invention also relates to the use of1,6-dimethyl-8β-hydroxymethyl-10α-methoxyergoline for preparing apharmaceutical which is of use in preventing and/or treating motorneuron diseases, in particular amyotrophic lateral sclerosis,progressive spinal muscular atrophy, infantile muscular atrophy andprimary lateral sclerosis.

[0035] 1,6-Dimethyl-8β-hydroxymethyl-10α-methoxy-ergoline can beprepared using the method described in French Patent No. 2,616,788.

[0036] The pharmaceuticals at least comprise1,6-dimethyl-8β-hydroxymethyl-10α-methoxyergoline, either in the purestate or in the form of a composition in which it is associated with anyother pharmaceutically compatible product, which may be inert orphysiologically active. The pharmaceuticals according to the inventioncan in particular be employed by the oral route or by the parenteralroute.

[0037] It is possible to use tablets, pills, powders (gelatin capsulesand tablets), oral lyophilisates (Lyoc^(R)) or granules as solidcompositions for oral administration. In these compositions, the activeprinciple is mixed with one or more inert diluents, such as starch,tartaric acid, gum arabic, sodium saccharin, vanillin, cellulose,sucrose, lactose or silica under a current of argon. These compositionscan also comprise substances other than diluents, for example one ormore lubricants such as magnesium stearate or talc, a dye, a coating(sugar-coated tablets) or a varnish.

[0038] It is possible to use pharmaceutically acceptable solutions,suspensions, emulsions, syrups and elixirs, containing inert diluentssuch as water, ethanol, glycerol, vegetable oils or paraffin oil, asliquid compositions for oral administration. These compositions cancomprise substances other than the diluents, for example wetting,sweetening, thickening, aromatizing or stabilizing products.

[0039] The sterile compositions for parenteral administration canpreferably be aqueous or nonaqueous solutions, suspensions or emulsions.It is possible to employ water, propylene glycol, a polyethylene glycol,vegetable oils, in particular olive oil, injectable organic esters, forexample ethyl oleate, or other suitable organic solvents, as solvent orvehicle. These compositions can also comprise adjuvants, in particularwetting, isotonizing, emulsifying, dispersing and stabilizing agents.Sterilization can be effected in a variety of ways, for example byasepticizing filtration, by incorporating sterilizing agents into thecomposition, by irradiation or by heating. The compositions can also beprepared in the form of sterile solid compositions which can bedissolved at the time of use in sterile water or any other injectablesterile medium.

FORMULATION EXAMPLES

[0040]

[0041] Capsule: 10 mg of1,6-dimethyl-8β-hydroxymethyl-10α-methoxyergoline and, as excipients,talc, lactose and magnesium stearate.

[0042] Oral lyophilizate: 10 mg of1,6-dimethyl-8β-hydroxymethyl-10α-methoxyergoline and, as excipients,tartaric acid, lactose, gum arabic, sodium saccharin and vanillin.

[0043] Powder for parenteral use: 10 mg of1,6-dimethyl-8β-hydroxymethyl-10α-methoxyergoline and, as excipients,tartaric acid and lactose.

[0044] The doses depend on the sought-after effect, on the duration ofthe treatment and on the administration route employed; they aregenerally between 30 and 200 mg per day by the oral route for an adult,with unit doses ranging from 5 to 50 mg of1,6-dimethyl-8β-hydroxymethyl-10α-methoxyergoline.

[0045] In a general manner, the doctor will determine the appropriatedosage according to the weight and all the other factors which arepeculiar to the subject to be treated.

[0046] The invention also relates to the method for preventing and/ortreating motor neuron diseases, in particular amyotrophic lateralsclerosis, progressive spinal muscular atrophy, infantile muscularatrophy and primary lateral sclerosis, which consists in administering1,6-dimethyl-8β-hydroxymethyl-10α-methoxyergoline to the patient.

1. A pharmaceutical composition comprising at least about 5 mg of1,6-dimethyl-8β-hydroxymethyl-10α-methoxyergoline or a pharmaceuticallyacceptable salt thereof, optionally in combination with apharmaceutically acceptable carrier useful in the treatment orprevention of motor neuron diseases.
 2. The composition of claim 1wherein the motor neuron disease is amyotrophic lateral sclerosis,progressive spinal muscular atrophy, infantile muscular atrophy orprimary lateral sclerosis.
 3. The composition of claim 2 wherein thedisease is amyotrophic lateral sclerosis.
 4. The composition of claim 2wherein the disease is progressive spinal muscular atrophy.
 5. Thecomposition of claim 2 wherein the disease is infantile muscularatrophy.
 6. The composition of claim 2 wherein the disease is primarylateral sclerosis.
 7. A pharmaceutical composition comprising from about5 to about 50 mg of 1,6-dimethyl-8β-hydroxymethyl-10α-methoxyergoline ora pharmaceutically acceptable salt thereof, optionally in combinationwith a pharmaceutically acceptable carrier useful in the treatment orprevention of motor neuron diseases.
 8. The composition of claim 7wherein the motor neuron disease is amyotrophic lateral sclerosis,progressive spinal muscular atrophy, infantile muscular atrophy orprimary lateral sclerosis.
 9. The composition of claim 7 wherein thedisease is amyotrophic lateral sclerosis.
 10. The composition of claim 7wherein the disease is progressive spinal muscular atrophy.
 11. Thecomposition of claim 7 wherein the disease is infantile muscularatrophy.
 12. The composition of claim 7 wherein the disease is primarylateral sclerosis.